EP1913151A1 - Oxidised phospholipids comprising a fluorophore moiety and use in the determination of the presence of enzymes having antiatherogenetic activity - Google Patents

Oxidised phospholipids comprising a fluorophore moiety and use in the determination of the presence of enzymes having antiatherogenetic activity

Info

Publication number
EP1913151A1
EP1913151A1 EP06760804A EP06760804A EP1913151A1 EP 1913151 A1 EP1913151 A1 EP 1913151A1 EP 06760804 A EP06760804 A EP 06760804A EP 06760804 A EP06760804 A EP 06760804A EP 1913151 A1 EP1913151 A1 EP 1913151A1
Authority
EP
European Patent Office
Prior art keywords
general formula
oxidized phospholipid
glycero
sample
enzymes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP06760804A
Other languages
German (de)
French (fr)
Other versions
EP1913151B1 (en
Inventor
Albin Hermetter
Michael Trenker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Technische Universitaet Graz
Original Assignee
Technische Universitaet Graz
Forschungsholding TU Graz GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Technische Universitaet Graz, Forschungsholding TU Graz GmbH filed Critical Technische Universitaet Graz
Publication of EP1913151A1 publication Critical patent/EP1913151A1/en
Application granted granted Critical
Publication of EP1913151B1 publication Critical patent/EP1913151B1/en
Not-in-force legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/10—Phosphatides, e.g. lecithin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/10—Phosphatides, e.g. lecithin
    • C07F9/106—Adducts, complexes, salts of phosphatides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6596—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having atoms other than oxygen, sulfur, selenium, tellurium, nitrogen or phosphorus as ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/323—Arteriosclerosis, Stenosis

Definitions

  • Oxidative modification of LDL is considered an important pathogenetic factor in atherosclerosis.
  • Studies from several laboratories have revealed that the biological effects triggered by mmLDL can largely be attributed to phospholipid oxidation products (Leitinger, N. et al. (1999) PNAS 96, 12010-12015; Watson, A. D. et al. (1995) J.Clin.Invest. 95, 774- 782; Leitinger, N. et al. (1997) Adv.Exp.Med.Biol 433, 379-382; Loidl, A. et al. (2003) J.Biol.Chem. 278, 32921-32928). Their increased levels in atherosclerotic plaques (Watson, A. D.
  • WO 01/75170 A a method of evaluating the risk for atherosclerosis is described, in which a biological sample comprising HDL is contacted with an oxidized phospholipid and the change in the amount of oxidized or non-oxidized phospholipid is measured, wherein the absence of change in the amount of oxidized phospholipid indicates a risk for atherosclerosis.
  • Oxidized phospholipids are involved in the pathogenesis of atherosclerosis. Oxidized phospholipids are further known to be hydrolytically cleaved by enzymes which are associated with lipoproteins and have antiatherogenetic activity, for example phospholipases or PAF acetylhydrolases.
  • their interaction with the enzymes should result in detectable products which allow to draw conclusions about the enzyme activity of the sample.
  • A O, C, NH, or S
  • B O, C, NH, or S
  • R 2 is selected from the group consisting of -CO-(CH 2 ) n -CH 3 ;
  • R 1 is selected from the group consisting of
  • R 1 is selected from the group consisting of
  • R 3 is selected from the group consisting of
  • a and/or B is oxygen.
  • X is a fluorophore selected from the group consisting of pyrene, perylene and nitrobenzaxadiazole, and preferably is pyrene.
  • X is a fluorophore selected from the group consisting of pyrene, perylene, borodiazaindacene (BODIP YTM), cyanine dye 2, cyanine dye 3, cyanine dye 5 and Alexa dyes.
  • the fluorescently labeled oxidized phospholipids according to the invention can be used as substrates for enzymes having antiatherogenetic activity.
  • the claimed compounds are particularly useful for the diagnostic determination of these enzymes in blood samples.
  • the fluorescently labeled cleavage products resulting from interaction of the oxidized phospholipids with the enzymes in question can be easily and sensitively determined by chromatographic analysis such as HPLC and thereby provide information about the enzyme activities present in the sample.
  • Another aspect of the invention thus refers to the use of the oxidized phospholipid according to the invention for determining the presence of enzymes having antiatherogenetic activity, preferably phospholipases or PAF-acetylhydrolases, in a sample.
  • a further aspect of the invention refers to a method for determining the presence of enzymes having antiatherogenetic activity in a sample comprising adding oxidized phospholipid according to the invention to the sample and subjecting the sample to chromatographic analysis, preferably HPLC.
  • PGPC (Fig. IA, 1) was synthesized according to a modified version of the procedure by Watson et al. (J.Biol.Chem. 272, 13597-13607, 1997) and Subbanagounder et at. ⁇ Free Radic.BiolMed. 28, 1751-1761, 2000).
  • PGPE (Fig. ⁇ A, 2) was obtained by transphosphatidylation of PGPC (90 mg, 148 ⁇ mol) (Fig. IA, 1) by PLD (phospholipase D, 29 units) in an emulsion of 1.4 ml of toluene and 4.3 ml of a 0.5 M sodium acetate buffer (pH 7.2) containing 0.5 M ethanolamine (Fig. IA, step 1). The triphasic system was stirred at 35 0 C over night. The reaction was monitored by TLC and quenched by addition of 4.3 ml of MeOH. The product was extracted with 43 ml of CHCl 3 /MeOH (2:1, v/v).
  • Alexa647-PGPE corresponds to BODIPY-PGPE (Fig. IA, 3) but contains Alexa647 instead of BODIPY at its head group. It was obtained from Alexa647-SE (Alexa Fluor 647 carboxylic acid succinimidyl ester, 0.5 mg, 0.4 ⁇ mol) and PGPE (0.68 mg, 1.2 ⁇ mol, 3 eq) (Fig. IA, 2) in 0.5 ml of CHCl 3 MeOH (2:1, v/v) after addition of triethylamine p.a. (5 ⁇ l, 36 ⁇ mol, 90 eq) followed by stirring at room temperature for 90 min. The reaction mixture was strictly protected from light.
  • the progress of the reaction was monitored by TLC (RP-18 F 2545 ; Merck, Darmstadt, Germany).
  • BODIPY-SE (1.00 mg, 2.57 ⁇ mol), triethylamine p.a. (10 ⁇ l, 72 ⁇ mol, 28 eq) and POPE (1- palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 5.53 mg, 7.70 ⁇ mol, 3 eq) were dissolved in 1 ml of CHCl 3 /MeOH (2:1, v/v). The mixture was stirred at room temperature for 60 min, then the solvent was removed under a stream of nitrogen. The white oily residue was dissolved in 500 ⁇ l of CHCl 3 /MeOH (2:1, v/v). The product was purified by preparative TLC.
  • the product was extracted from the aqueous solution with CHCl 3 /MeOH (2:1, v/v) (3 x 5 ml). The combined organic fractions were evaporated and the residual water was removed under high vacuum leading to 50 mg of a mixture of l-(10- pyrenedecanoyl)-sn-glycero-3-phosphocholine (Fig. 3, 12) and free pyrenedecanoic acid. The latter one was removed by trituration with diethylether.
  • l-tlO-Pyrenedecanoyty-s/z-glycero-S-phosphocholine (10 mg, 16 ⁇ mol) (Fig. 3, 12) was acylated with 5,5-dimethoxypentanoic acid (acid "5") in 10 ml of dichloromethane containing DCC (200 mg, 1.0 mmol, 59 eq) and DMAP (200 nig, 1.6 mmol, 100 eq). The reaction mixture was stirred at room temperature over night (Fig. 3, step 2b). The progress of the reaction was monitored by TLC until the reaction was stopped by addition of 6 ml of MeOH.
  • PEG poly(ethylene glycol)s
  • TFA trifluoroacetic acid
  • the spectra of 50- 100 shots were averaged to improve the signal-to-noise ratio. All m/z values discussed in this work correspond to the most intense peak of any isotope distribution.
  • the mass spectra of BODIPY-POVPE, BODPY-PGPE and BODIPY-POPE are shown in Fig. 2.
  • the mass spectra of PyrGPC and PyrOVPC are shown in Fig. 4.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Endocrinology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to oxidized phospholipids having one of the general formulas (I) or (II) wherein A═O, C, NH, or S; B═O, C, NH, or S; and R2 is selected from the group consisting of —CO—(CH2)n—CH3; —CO—(CH2)n—CHO; and —CO—(CH2)n—COOH, with n=3-7, with the proviso that in general formula (I), R1 is selected from the group consisting of —CH2—(CH2)n—X; and —CO—(CH2)n—X with n=5-11, wherein X is a fluorophore; and in general formula (II), R1 is selected from the group consisting of —CH═CH—(CH2)n—CH3 with n=9-15; —(CH2)n—CH3 with n=11-17; and —CO—(CH2)n—CH3 with n=10—16; and R3 is selected from the group consisting of —CO—(CH2)n—X; and —SO2—(CH2)n—X, with n=0-5, wherein X is a fluorophore.

Description

OXIDISED PHOSPHOLIPIDS COMPRISING A FLUOROPHORE MOIETY AND USE IN THE DETERMINATION OF THE PRESENCE OF ENZYMES HAVING ANTIATHEROGENETIC ACTIVITY
Oxidative modification of LDL is considered an important pathogenetic factor in atherosclerosis. Studies from several laboratories have revealed that the biological effects triggered by mmLDL can largely be attributed to phospholipid oxidation products (Leitinger, N. et al. (1999) PNAS 96, 12010-12015; Watson, A. D. et al. (1995) J.Clin.Invest. 95, 774- 782; Leitinger, N. et al. (1997) Adv.Exp.Med.Biol 433, 379-382; Loidl, A. et al. (2003) J.Biol.Chem. 278, 32921-32928). Their increased levels in atherosclerotic plaques (Watson, A. D. et al.(1997) J.Biol.Chem. 272, 13597-13607; Itabe, H. et al. (1994) J.Biol.Chem. 269, 15274-15279) and the elevated antibody titers against oxidized phospholipids in humans and mice with lesions (Horkko, S. et al. (1999) J.Clin.Invest. 103, 117-128; Palinski, W. et al. (1995) Arterioscler.Thromb. Vasc.Biol. 15, 1569-1576; Palinski, W. et al. (1996) J.Clin.Invest. 98, 800-814) attract attention to the pathological relevance of these molecules.
A rapidly growing interest has been focused on two major representatives in the series of homologous oxidized phospholipids, namely l-palmitoyl-2-glutaroyl-5«-glycero-3- phosphocholine (PGPC) and l-palmitoyl-2-(5-oxovaleroyl)-OT-glycero-3-phosphocholine (POVPC). Their importance is stressed by the finding that they selectively activate processes in vascular wall cells that may contribute to the pathogenesis of atherosclerosis as well as other chronic inflammatory diseases. Both lipids were demonstrated to be 3- to 6-fold enriched in rabbit atherosclerotic lesions corresponding to approximately 62 and 116 ng/mg of aorta wet weight PGPC and POVPC, respectively ( Subbanagounder, G. et al. (2000) Arterioscler.Thromb. Vasc.Biol. 20, 2248-2254).
In WO 01/75170 A a method of evaluating the risk for atherosclerosis is described, in which a biological sample comprising HDL is contacted with an oxidized phospholipid and the change in the amount of oxidized or non-oxidized phospholipid is measured, wherein the absence of change in the amount of oxidized phospholipid indicates a risk for atherosclerosis.
As mentioned above, oxidized phospholipids are involved in the pathogenesis of atherosclerosis. Oxidized phospholipids are further known to be hydrolytically cleaved by enzymes which are associated with lipoproteins and have antiatherogenetic activity, for example phospholipases or PAF acetylhydrolases.
It is the object of the present invention to provide compounds which can be used to gather information about the activity of antiatherogenetic enzymes in a sample. In particular, their interaction with the enzymes should result in detectable products which allow to draw conclusions about the enzyme activity of the sample.
This object is achieved by an oxidized phospholipid having one of the general formulas I or II
wherein
A = O, C, NH, or S;
B = O, C, NH, or S; and
R2 is selected from the group consisting of -CO-(CH2)n-CH3;
-CO-(CH2)n-CHO; and -CO-(CH2)n-COOH, with n = 3-7, with the proviso that in general formula I, R1 is selected from the group consisting of
-CH2-(CH2)n-X; and
-CO-(CH2)n-X with n = 5-11, wherein X is a fluorophore; and in general formula II, R1 is selected from the group consisting of
-CH=CH-(CH2)n-CH3 with n = 9-15;
-(CH2)n-CH3 with n = 11-17; and
-CO-(CH2)n-CH3 with n = 10-16; and R3 is selected from the group consisting of
-CO-(CH2)n-X; and
-SO2-(CH2)n-X, with n = 0-5, wherein X is a fluorophore. According to a preferred embodiment, A and/or B is oxygen.
According to another preferred embodiment, in general formula I, X is a fluorophore selected from the group consisting of pyrene, perylene and nitrobenzaxadiazole, and preferably is pyrene.
According to another preferred embodiment, in general formula π, X is a fluorophore selected from the group consisting of pyrene, perylene, borodiazaindacene (BODIP Y™), cyanine dye 2, cyanine dye 3, cyanine dye 5 and Alexa dyes.
Preferred embodiments of oxidized phospholipids according to general formula I are the following compounds:
• 1 -( 10-pyrenedecanoyl)-2-glutaroyl-5«-glycero-3 -phosphocholine (PyrGPC)
• 1 -(10-pyrenedecanoyl)-2-(5-oxovaleroyl)-5i«-glycero-3-phosphocholine (PyrOVPC)
Preferred embodiments of oxidized phospholipids according to general formula II are the following compounds:
• l-palmitoyl-2-glutaroyl-5r«-glycero-3-phospho-N-(3-[4,4-difluoro-4-bora-3a,4a-diaza- s-indacene]-propionyl)-ethanolamine (BODIPY-PGPE)
• 1 -pahnitoyl-2-glutaroyl-5'77-glycero-3 -phospho-N-(Alexa Fluor 647-carbonyl)- ethanolamine (Alexa647-PGPE)
• l-palmitoyl-2-oleoyl-5Η-glycero-3-phospho-iV-(3-[4,4-difluoro-4-bora-3a,4a-diaza-5- indacene]-propionyl)-ethanolamine (BODIPY-POPE)
• l-pahnitoyl-2-(5-oxovaleroyl)-5n-glycero-3-phospho-iV-(3-[4,4-difluoro-4-bora-3a,4a- diaza-5-indacene]-propionyl)-ethanolamine (BODPY-POVPE)
It has been found that the fluorescently labeled oxidized phospholipids according to the invention can be used as substrates for enzymes having antiatherogenetic activity. The claimed compounds are particularly useful for the diagnostic determination of these enzymes in blood samples. The fluorescently labeled cleavage products resulting from interaction of the oxidized phospholipids with the enzymes in question can be easily and sensitively determined by chromatographic analysis such as HPLC and thereby provide information about the enzyme activities present in the sample. Another aspect of the invention thus refers to the use of the oxidized phospholipid according to the invention for determining the presence of enzymes having antiatherogenetic activity, preferably phospholipases or PAF-acetylhydrolases, in a sample.
A further aspect of the invention refers to a method for determining the presence of enzymes having antiatherogenetic activity in a sample comprising adding oxidized phospholipid according to the invention to the sample and subjecting the sample to chromatographic analysis, preferably HPLC.
The present invention will be explained in more detail by way of the following examples and the attached drawings.
Examples:
Synthesis of fluorescent oxidized phospholipids
Preparation of l-palmitoyl-2-gIutaroyl-sw-glycero-3-phospho-iV-(3-[4,4-difluoro-4-bora- 3a,4a-diaza-s-indacene]-propionyl)-ethanolamme (BODIPY-PGPE)
1. Synthesis of l-palmitoyl^-glutaroyl-sn-glycero-S-phosphocholine (PGPC)
PGPC (Fig. IA, 1) was synthesized according to a modified version of the procedure by Watson et al. (J.Biol.Chem. 272, 13597-13607, 1997) and Subbanagounder et at. {Free Radic.BiolMed. 28, 1751-1761, 2000). A solution of dry l-palmitoyl-sn-glycero-3- phosphocholine (62 mg, 125 μmol), dry glutaric anhydride (70 mg, 613 μmol, 5 eq) and DMAP (p-(N,7V'-dimethylamino)pyridine, 75 mg, 614 μmol, 5 eq) in 6 ml of anhydrous dichloromethane was magnetically stirred over night at 350C. The reaction was monitored by TLC with CHCl3/Me0H/25%NH3 (65:35:5, v/v/v) as a developing system, and quenched by addition of 3 ml of MeOH. The resulting mixture was washed once with 1.8 ml of MeOHTH2O (1:1, v/v). After evaporation of the organic phase, excess DMAP was removed by trituration with 4 ml of diethylether. Removal of the supernatant afforded 32 mg of PGPC (42%, Rf= 0.05).
2. Synthesis of l-palmitoyl-2-glutaroyl-sn-glycero-3-phosphoethanolamine (PGPE)
PGPE (Fig. \A, 2) was obtained by transphosphatidylation of PGPC (90 mg, 148 μmol) (Fig. IA, 1) by PLD (phospholipase D, 29 units) in an emulsion of 1.4 ml of toluene and 4.3 ml of a 0.5 M sodium acetate buffer (pH 7.2) containing 0.5 M ethanolamine (Fig. IA, step 1). The triphasic system was stirred at 350C over night. The reaction was monitored by TLC and quenched by addition of 4.3 ml of MeOH. The product was extracted with 43 ml of CHCl3/MeOH (2:1, v/v). The organic phase was washed twice with 11 ml of MeOH/H2O (1:1, v/v) to remove excess ethanolamine, evaporated and subjected to preparative TLC. The product was scraped off and eluted three times from the silica gel with CHCl3ZMeOH (1 :4, v/v). Evaporation of the combined extracts yielded the desired product (30.3 mg, 36%, Rf = 0.30).
3. Synthesis of l-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-(3-[4,4-difluoro-4-bora- 3a,4a-diaza-s-indacene]-propionyl)-ethanolarnine (BODlPY-PGPE)
To a magnetically stirred solution of BODIPY-SE (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-propionic acid succinimidyl ester, 0.96 mg, 2.5 μmol) in 1 ml of CHCl3/MeOH (2:1, v/v) were added PGPE (4.4 mg, 7.8 μmol, 3 eq) (Fig. IA, 2) and triethylamine p. a. (10 μl, 72 μmol, 30 eq) (Fig. IA, step 2). Then the flask was flushed with nitrogen, protected from light, and the resulting solution was stirred at room temperature for 1 h. The progress of the reaction was monitored by TLC. The solvent was removed under a nitrogen stream until a volume of 400 μl was reached, from which the lipid was purified by preparative TLC. The desired compound was visualized under UV light, scraped off the TLC plate and eluted three times from the silica gel with CHCl3/MeOH (1:4, v/v). The solvent was removed from the combined extracts by rotary evaporation to afford BODIP Y-PGPE (1.77 mg, 85%, Rf= 0.28) (Fig. IA, 3).
Preparation of l-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-(Alexa647-carbonyl)- ethanolamine (Alexa647-PGPE)
Alexa647-PGPE corresponds to BODIPY-PGPE (Fig. IA, 3) but contains Alexa647 instead of BODIPY at its head group. It was obtained from Alexa647-SE (Alexa Fluor 647 carboxylic acid succinimidyl ester, 0.5 mg, 0.4 μmol) and PGPE (0.68 mg, 1.2 μmol, 3 eq) (Fig. IA, 2) in 0.5 ml of CHCl3MeOH (2:1, v/v) after addition of triethylamine p.a. (5 μl, 36 μmol, 90 eq) followed by stirring at room temperature for 90 min. The reaction mixture was strictly protected from light. The progress of the reaction was monitored by TLC (RP-18 F2545; Merck, Darmstadt, Germany). The product was purified by preparative TLC with H2O/EtOH/n-propanol (20:57:23, v/v/v) as a developing system, scraped off and eluted twice with CHCl3/MeOH (1:1, v/v) and once with MeOH. Removal of the solvent under a stream of nitrogen provided Alexa647-PGPE (0.26 μmol, 65%, Rf = 0.80). Preparation of l-palmitoyl-2-(5-oxovaIeroyl)-sn-glycero-3-phospho-N-(3-[4,4-difluoro-4- bora-3a,4a-diaza-s-indacene]-propionyl)-ethanolamine (BODIPY-POVPE)
1. Synthesis of l-palmitoyl-2-(5-dimethoxypentan.oyl)-sn-glycero-3-phosphoethanolamine
A solution of 5,5-dimethoxypentanoic acid methyl ester (220 mg, 1.25 mniol) (Fig. IB, 4) and sodium hydroxide (250 mg, 6.25 mmol, 5 eq) in 5 ml of H2OMeOHzTHF (2:5:3, v/v/v) was stirred at room temperature for 90 min (Fig. IB, step 1). After cooling down to 0°C, the reaction mixture was acidified to pH 2.1 by subsequent addition of 6 ml of 1 N and appropriate amounts of 0.1 N HCl and then extracted with dichloromethane (3 x 15 ml). The combined organic extracts were washed with water (2 x 10 ml) and dried over Na2SO4. The solvent was removed under reduced pressure except for 10-15 ml containing the desired product (Rf = 0.35) (Fig. IB, 5), which was immediately used for the acylation reaction without further purification. l-Palmitoyl-s«-glycero-3-phosphocholine (209 mg, 0.42 mmol) (Fig. IB, 6), DCC (N.N'-dicyclohexylcarbodiimide, 270 mg, 1.3 mmol, 3 eq) and DMAP (160 mg, 1.3 mmol, 3 eq) were added to this solution of 5,5-dimethoxypentanoic acid (Fig. IB, step X). The mixture was stirred under nitrogen at room temperature over night. The reaction was monitored by TLC. After addition of 6 ml of MeOH, the organic solution was washed twice with MeOH/H2O (1:1, v/v). The solvent was removed under vacuum, and traces of water were evaporated after addition of benzene/EtOH (3:2, v/v). The oily residue was flash chromatographed on 12 g silica gel with CHCl3/Me0H/25%NH3 (65:35:5, v/v/v) as a solvent to give l-palmitoyl-2-(5-dimethoxypentanoyl)-1sin-glycero-3-phosphocholine (245 mg, 91%, Rf = 0.14) (Fig. IB, 7). The ethanolamine analog (Fig. IB, 8) was obtained by transphosphatidylation of the choline lipid as described previously for the conversion of PGPC to PGPE (compare Fig. IA, step 1) (yield: 6.2 mg, 66%, Rf = 0.24 using CHCl3/MeOH/25%NH3, 65:35:5, v/v/v as a developing solvent) (Fig. IB, step 3).
2. Synthesis of l-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phospho-N-(3-[4,4-difluoro-4- bora-3 a,4a-diaza-5-indacene] -propionyl)-ethanolamine (BODIP Y-POVPE)
Triethylatnine p. a. (10 μl, 72 μmol, 14 eq) was added to a solution of l-palmitoyl-2-(5- dimemoxypentanoyty-sn-glycero-S-phosphoethanolamine (3.0 mg, 5.0 μmol) (Fig. IB, 8) and BODIPY-SE (1.95 mg, 5.0 μmol, 1 eq) in 1 ml of CHCl3/MeOH (2:1, v/v) (Fig. IB, step 4). The reaction mixture was stirred at room temperature for 80 min. The solvent was removed under vacuum yielding l-palmitoyl-2-dimemoxypentanoyl-sra-glycero-3-phospho-N-(3- BODIPY-propionyl)-ethanolamine (Fig. IB, 9) that was suitable for the next reaction without further purification. Release of the desired product was accomplished by acetal cleavage of this stable precursor (2.0 mg, 2.3 μmol) with 400 μl of THF/HC1 (1 N) (Fig. IB, step 5). After only 2 min, the reaction mixture was neutralized with NaHCO3 followed by extraction of the product with 1.2 ml of CHCl3/MeOH (2:1, v/v). The organic phase was washed twice with 300 μl of water, dried over Na2SO4 and evaporated under reduced pressure leading to 1.65 mg of BODlP Y-POVPE (87%, Rf = 0.22 using CHCl3/MeOH/H20, 15:5:0.1, v/v/v as a developing system) (Fig. IB, 10).
Preparation of l-palmitoyl-2-oIeoyl-sn-glycero-3-phospho-N-(3-[4,4-difluoro-4-bora- 3a,4a-diaza-s-indacene]-propionyl)-ethanolamine (BODIPY-POPE)
BODIPY-SE (1.00 mg, 2.57 μmol), triethylamine p.a. (10 μl, 72 μmol, 28 eq) and POPE (1- palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 5.53 mg, 7.70 μmol, 3 eq) were dissolved in 1 ml of CHCl3/MeOH (2:1, v/v). The mixture was stirred at room temperature for 60 min, then the solvent was removed under a stream of nitrogen. The white oily residue was dissolved in 500 μl of CHCl3/MeOH (2:1, v/v). The product was purified by preparative TLC. The fluorescent band containing the product was scraped off the plate and eluted three times with CHCl3/MeOH (1:4, v/v). The combined extracts were evaporated to deliver the desired product (1.31 mg, 51%, Rf = 0.59).
Synthesis of l-(10-pyrenedecanoyl)-2-glutaroyl-s»-glycero-3-phosphocholine (PyrGPC)
To a magnetically stirred emulsion of l,2-bis(10-pyrenedecanoyl)-5«-glycero-3- phosphocholine (45 mg, 47 μmol) (Fig. 3, 11) in a mixture of 3 ml of 0.1 M Tris-HCl buffer (pH 8) containing 0.1 M CaCl2 and 3 ml of diethylether (peroxide-free) were added 50 units OfPLA2 (Naja naja venom) (Fig. 3, step 1). The reaction mixture was stirred overnight at 35- 400C. After removing the diethylether, the product was extracted from the aqueous solution with CHCl3/MeOH (2:1, v/v) (3 x 5 ml). The combined organic fractions were evaporated and the residual water was removed under high vacuum leading to 50 mg of a mixture of l-(10- pyrenedecanoyl)-sn-glycero-3-phosphocholine (Fig. 3, 12) and free pyrenedecanoic acid. The latter one was removed by trituration with diethylether. After removal of the solvent under vacuum, 25 mg of the pure lysophospholipid were obtained (88%, Rf = 0.12 in CHCl3MeOHZAcOHZH2O, 50:30:10:5, VNMY as a developing system). Glutaric anhydride (12 mg, 105 μmol, 11 eq) and anhydrous DMAP (4 mg, 33 μmol, 3 eq) were added to a solution of the lysophospholipid (6.0 mg, 9.8 μmol) in 3 ml of anhydrous dichloromethane (Fig. 3, step 2a). The reaction was stirred over night at 35-40°C. Flash chromatography of the crude product on 10 g of silica gel with CHCl3/MeOH/H2O (65:25:4), vZv/v) led to PyrGPC (2.2 mg, 31%, Rf = 0.18 in CHCl3ZMeOHZAcOHZH2O, 50:30:10:5, vhhh as a developing system) (Fig. 3, 14).
Synthesis of l-(10-pyrenedecanoyI)-2-(5-oxovaIeroyl)-s«-glycero-3-phosphocholinie (PyrOVPC)
l-tlO-Pyrenedecanoyty-s/z-glycero-S-phosphocholine (10 mg, 16 μmol) (Fig. 3, 12) was acylated with 5,5-dimethoxypentanoic acid (acid "5") in 10 ml of dichloromethane containing DCC (200 mg, 1.0 mmol, 59 eq) and DMAP (200 nig, 1.6 mmol, 100 eq). The reaction mixture was stirred at room temperature over night (Fig. 3, step 2b). The progress of the reaction was monitored by TLC until the reaction was stopped by addition of 6 ml of MeOH. The resultant solution was washed twice with MeOHZH2O (1:1, vZv), and the solvent was removed under reduced pressure. The crude product was purified by preparative TLC, scraped off and eluted five times with 4 ml of CHCl3ZMeOH (1 :4, vZv). Residual silica gel was removed by washing the combined organic fractions (concentrated to a volume of 5 ml) with 1 ml OfMeOHZH2O (1:1, vZv). Evaporation of the solvent yielded 6 mg of l-(10- pyrenedecanoyl)-2-(5-dimethoxypentanoyl)-57ϊ-glycero-3-phosphocholine (49%, Rf = 0.17) (Fig. 3, 15). Deprotection of intermediate 15 (5.6 mg, 7.4 μmol) was accomplished by acetal cleavage with THFZHCl (1 N), followed by neutralization with NaHCO3 and extraction of the product with CHCl3ZMeOH (2:1, vZv). The organic phase was washed twice, dried over Na2SO4 and evaporated under reduced pressure leading to 1.1 mg of PyrOVPC (21%, Rf = 0.05) (Fig. 3, step 3).
Mass Spectrometry
Matrix assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectra were performed on a Micromass TofSpec2E equipped with a nitrogen laser (λ = 337 nm, operated at 5 Hz) and a time-lag focusing unit. Ions were generated by irradiation just above the threshold laser power. The spectra were recorded in the reflectron mode with an acceleration voltage of 20 kV and externally calibrated with a suitable mixture of poly(ethylene glycol)s (PEG). Samples were typically prepared by mixing solutions of the matrix (2,5- dihydroxybenzoic acid, c = 10 mg/ml, CH3CNZ0.1% trifluoroacetic acid (TFA), 70:30, vZv), the analyte (c = 0.01-1 mgZml, CHCl3ZMeOH, 2:1, vZv) and NaTFA (c = 1 mgZml, CH3CNZH2O, 70:30, vZv) in a ratio of 10:1:0.5 (v/v/v). A 0.5 μl aliquot of the mixture was deposited on the sample plate (stainless steel) and allowed to dry under air. The spectra of 50- 100 shots were averaged to improve the signal-to-noise ratio. All m/z values discussed in this work correspond to the most intense peak of any isotope distribution. The mass spectra of BODIPY-POVPE, BODPY-PGPE and BODIPY-POPE are shown in Fig. 2. The mass spectra of PyrGPC and PyrOVPC are shown in Fig. 4.

Claims

1. An oxidized phospholipid having one of the general formulas I or II
wherein
A = O, C, NH, or S;
B = O, C5 NH, or S; and
R2 is selected from the group consisting of -CO-(CH2)n-CH3;
-CO-(CH2)n-CHO; and -CO-(CH2)n-COOH, with n = 3-7, with the proviso that in general formula I, R1 is selected from the group consisting of
-CH2-(CH2)n-X; and
-C0-(CH2)n-X with n = 5-11 , wherein X is a fluorophore; and in general formula II, R1 is selected from the group consisting of
-CH=CH-(CH2)n-CH3 with n = 9-15;
-(CH2)n-CH3 with n = 11-17; and
-CO-(CH2)n-CH3 with n = 10-16; and R3 is selected from the group consisting of
-C0-(CH2)n-X; and
-SO2-(CH2)n-X, with n = 0-5, wherein X is a fluorophore.
2. The oxidized phospholipid of general formula I according to claim 1, wherein A and/or B is oxygen.
3. The oxidized phospholipid of general formula I according to claim 1 or 2, wherein X is a fluorophore selected from the group consisting of pyrene, perylene and nitrobenzaxadiazole, and preferably is pyrene.
4. The oxidized phospholipid of general formula I according to any of claims 1 to 3, characterized as ^(lO-pyrenedecanoy^-l-glutaroyl-sπ-glycero-S-phosphocholine (PyrGPC).
5. The oxidized phospholipid of general formula I according to any of claims 1 to 3, characterized as l-(10-pyrenedecanoyl)-2-(5-oxovaleroyl)-5R-glycero-3-phosphocholine (PyrOVPC).
6. Use of the oxidized phospholipid of general formula I according to any of claims 1 to 5 for determining the presence of enzymes having antiatherogenetic activity, preferably phospholipases or PAF-acetylhydrolases, in a sample.
7. A method for determining the presence of enzymes having antiatherogenetic activity in a sample comprising adding oxidized phospholipid of general formula I according to any of claims 1 to 5 to the sample and subjecting the sample to chromatographic analysis, preferably HPLC.
8. The oxidized phospholipid of general formula II according to claim 1 , wherein A and/or B is oxygen.
9. The oxidized phospholipid of general formula II according to claim 1 or 8, wherein X is a fluorophore selected from the group consisting of pyrene, perylene, borodiazaindacene (BODIP Y™), cyanine dye 2, cyanine dye 3, cyanine dye 5 and Alexa dyes.
10. The oxidized phospholipid of general formula II according to any of claims 1 , 8 or 9, characterized as l-pahnitoyl-2-glutaroyl-5fn-glycero-3-phospho-JV-(3-[4,4-difiuoro-4-bora- 3a,4a-diaza-s-indacene]~propionyl)-ethanolamine (BODIPY-PGPE).
11. The oxidized phospholipid of general formula II according to any of claims 1 , 8 or 9, characterized as l-palmitoyl-2-glutaroyl-5n-glycero-3-phospho-N-(Alexa Fluor 647- carbonyl)-ethanolamine (Alexa647-PGPE).
12. The oxidized phospholipid of general formula II according to any of claims 1, 8 or 9, characterized as 1 -palmitoyl-2-oleoyl-5«-glycero-3-phospho-N-(3-[4,4-difluoro-4-bora-3a,4a- diaza-s-indacene]-propionyl)-ethanolamine (BODIPY-POPE).
13. The oxidized phospholipid of general formula II according to any of claims 1, 8 or 9, characterized as l-palmitoyl-2-(5-oxovaleroyl)-5r«-glycero-3-phospho-N-(3-[4,4-difluoro-4- bora-3a,4a-diaza-5-indacene]-propionyl)-ethanolamine (BODIPY-POVPE).
14. Use of the oxidized phospholipid of general formula II according to any of claims 1, 8 to 13 for determining the presence of enzymes having antiatherogenetic activity, preferably phospholipases or PAF-acetylhydrolases, in a sample.
15. A method for determining the presence of enzymes having antiatherogenetic activity in a sample comprising adding oxidized phospholipid of general formula II according to any of claims 1, 8 to 13 to the sample and subjecting the sample to chromatographic analysis, preferably HPLC.
EP06760804A 2005-07-27 2006-07-26 Oxidised phospholipids comprising a fluorophore moiety and use in the determination of the presence of enzymes having antiatherogenetic activity Not-in-force EP1913151B1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AT12602005 2005-07-27
AT18262005 2005-11-08
PCT/AT2006/000317 WO2007012100A1 (en) 2005-07-27 2006-07-26 Oxidised phospholipids comprising a fluorophore moiety and use in the determination of the presence of enzymes having antiatherogenetic activity

Publications (2)

Publication Number Publication Date
EP1913151A1 true EP1913151A1 (en) 2008-04-23
EP1913151B1 EP1913151B1 (en) 2011-12-07

Family

ID=37401640

Family Applications (1)

Application Number Title Priority Date Filing Date
EP06760804A Not-in-force EP1913151B1 (en) 2005-07-27 2006-07-26 Oxidised phospholipids comprising a fluorophore moiety and use in the determination of the presence of enzymes having antiatherogenetic activity

Country Status (8)

Country Link
US (1) US7906674B2 (en)
EP (1) EP1913151B1 (en)
JP (1) JP5276981B2 (en)
KR (1) KR101299614B1 (en)
AT (1) ATE536423T1 (en)
AU (1) AU2006274477B2 (en)
CA (1) CA2654743C (en)
WO (1) WO2007012100A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107434814B (en) * 2017-07-14 2019-05-03 中国科学院化学研究所 Modified conjugated polymer and its preparation method and application

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FI63064C (en) * 1980-04-09 1983-04-11 Ksv Chemicals Oy FOERFARANDE FOER FLUOROMETRISK BESTAEMNING AV AKTIVITETEN AV FETTSPJAELKANDE ENZYMER OCH MEDEL FOER ATT GENOMFOERA FOERFARANDET
US7122344B2 (en) * 1997-05-28 2006-10-17 Claragen, Inc. Methods for the production of purified recombinant human uteroglobin for the treatment of inflammatory and fibrotic conditions
JP4220603B2 (en) 1998-12-02 2009-02-04 アルフレッサファーマ株式会社 Method for measuring platelet activating factor acetylhydrolase activity
ES2250378T3 (en) * 2000-03-31 2006-04-16 The Regents Of The University Of California FUNCTIONAL TEST OF HIGH DENSITY LIPOPROTEIN.
EP1318154B1 (en) 2001-12-07 2004-06-16 SIRS-Lab GmbH Compounds for determination of the activity of phospholipase A2
WO2005005977A2 (en) * 2003-06-30 2005-01-20 Applera Corporation Fluorescent phospholipase assays and compositions

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2007012100A1 *

Also Published As

Publication number Publication date
CA2654743A1 (en) 2007-02-01
CA2654743C (en) 2015-04-07
JP2009505956A (en) 2009-02-12
KR20080057228A (en) 2008-06-24
EP1913151B1 (en) 2011-12-07
KR101299614B1 (en) 2013-08-23
WO2007012100A1 (en) 2007-02-01
AU2006274477A1 (en) 2007-02-01
AU2006274477B2 (en) 2011-05-12
US7906674B2 (en) 2011-03-15
JP5276981B2 (en) 2013-08-28
US20090130648A1 (en) 2009-05-21
ATE536423T1 (en) 2011-12-15

Similar Documents

Publication Publication Date Title
Reis et al. Chemistry of phospholipid oxidation
EP3063288B1 (en) A method for labeling specifically living bacteria comprising the use of modified monosaccharide compounds
Sidhu et al. N-acyl-O-phosphocholineserines: Structures of a novel class of lipids that are biomarkers for Niemann-Pick C1 disease
Harrison et al. Analysis of oxidized glycerophosphocholine lipids using electrospray ionization mass spectrometry and microderivatization techniques
Orr et al. Synthesis of the diastereoisomers of 1, 2-dipalmitoyl-sn-glycero-3-thiophosphorylethanolamine and their stereospecific hydrolysis by phospholipases A2 and C
Bonsen et al. Synthesis and enzymic hydrolysis of an O-alanyl ester of phosphatidyl glycerol
Polyakova et al. New GM1 ganglioside derivatives for selective single and double labelling of the natural glycosphingolipid skeleton
EP1913151B1 (en) Oxidised phospholipids comprising a fluorophore moiety and use in the determination of the presence of enzymes having antiatherogenetic activity
AU708266B2 (en) 1,2 chemiluminescent dioxetanes of improved performance
Browning et al. Synthesis of specifically deuterated saturated and unsaturated phosphatidylserines
JP4150675B2 (en) Compound for measuring the activity of phospholipase A2
Kim et al. Synthesis of 1-lyso-2-palmitoyl-rac-glycero-3-phosphocholine and its regioisomers and structural elucidation by NMR spectroscopy and FAB tandem mass spectrometry
MX2008001391A (en) Oxidised phospholipids comprising a fluorophore moiety and use in the determination of the presence of enzymes having antiatherogenetic activity
Endo et al. Synthesis and physical properties of phosphatidylcholines containing ω-cyclohexyl fatty acyl chains
Antonopoulou et al. Isolation and structural elucidation of biologically active phospholipids from Scytonema julianum (cyanobacteria)
US7772402B2 (en) Optically detectable organophosphonates
Huang et al. Incorporation of a fluorous diazirine group into phosphatidylinositol 4, 5-bisphosphate to illustrate its interaction with ADP-ribosylation factor 1
Schmitz et al. 1-trans-Parinaroyl phospholipids: synthesis and fast atom bombardment/electron impact mass spectrometric characterization
WO2019135386A1 (en) Deuterated phospholipid and method for producing same
Erukulla et al. Three-step synthesis of platelet-activating factor from chiral glycidol via regioselective monophosphitylation of 1-O-hexadecyl-sn-glycerol
WO2007145361A1 (en) Lipid antigen detection method using mass spectrometer
PHOSPHORAMIDITE STEREOSPECIFIC SYNTHESIS OF PHOSPHATIDYL GLYCEROL USING A CYANOETHYL PHOSPHORAMIDITE PRECURSOR
EP3170832A1 (en) Method of preparation of 6-azido-2,4-diacetamido-2,4,6-trideoxy-d-mannose
CA2231199C (en) 1,2 chemiluminescent dioxetanes of improved performance
Zaikin et al. Specific electron ionization-induced fragmentation of secondary alcohol methoxyacetates

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20080227

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

17Q First examination report despatched

Effective date: 20090623

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

DAX Request for extension of the european patent (deleted)
REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602006026266

Country of ref document: DE

Effective date: 20120315

REG Reference to a national code

Ref country code: NL

Ref legal event code: VDEP

Effective date: 20111207

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: KIRKER & CIE S.A.

RAP2 Party data changed (patent owner data changed or rights of a patent transferred)

Owner name: TECHNISCHE UNIVERSITAET GRAZ

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

LTIE Lt: invalidation of european patent or patent extension

Effective date: 20111207

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120308

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

REG Reference to a national code

Ref country code: CH

Ref legal event code: PUEA

Owner name: TECHNISCHE UNIVERSITAET GRAZ

Free format text: TECHNISCHE UNIVERSITAET GRAZ#RECHBAUERSTRASSE 12#8010 GRAZ (AT) $ FORSCHUNGSHOLDING TU GRAZ GMBH#RECHBAUERSTRASSE 12#8010 GRAZ (AT) -TRANSFER TO- TECHNISCHE UNIVERSITAET GRAZ#RECHBAUERSTRASSE 12#8010 GRAZ (AT)

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120307

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120407

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

REG Reference to a national code

Ref country code: GB

Ref legal event code: 732E

Free format text: REGISTERED BETWEEN 20120726 AND 20120801

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120409

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602006026266

Country of ref document: DE

REG Reference to a national code

Ref country code: DE

Ref legal event code: R082

Ref document number: 602006026266

Country of ref document: DE

Representative=s name: MITSCHERLICH & PARTNER PATENT- UND RECHTSANWAE, DE

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

26N No opposition filed

Effective date: 20120910

REG Reference to a national code

Ref country code: DE

Ref legal event code: R081

Ref document number: 602006026266

Country of ref document: DE

Owner name: TECHNISCHE UNIVERSITAET GRAZ, AT

Free format text: FORMER OWNER: TECHNISCHE UNIVERSITAET GRAZ, FORSCHUNGSHOLDING TU GRAZ GMBH, , AT

Effective date: 20120924

Ref country code: DE

Ref legal event code: R082

Ref document number: 602006026266

Country of ref document: DE

Representative=s name: MITSCHERLICH & PARTNER PATENT- UND RECHTSANWAE, DE

Effective date: 20120924

Ref country code: DE

Ref legal event code: R082

Ref document number: 602006026266

Country of ref document: DE

Representative=s name: MITSCHERLICH, PATENT- UND RECHTSANWAELTE, PART, DE

Effective date: 20120924

Ref country code: DE

Ref legal event code: R082

Ref document number: 602006026266

Country of ref document: DE

Representative=s name: MITSCHERLICH, PATENT- UND RECHTSANWAELTE PARTM, DE

Effective date: 20120924

Ref country code: DE

Ref legal event code: R081

Ref document number: 602006026266

Country of ref document: DE

Owner name: TECHNISCHE UNIVERSITAET GRAZ, AT

Free format text: FORMER OWNERS: TECHNISCHE UNIVERSITAET GRAZ, GRAZ, AT; FORSCHUNGSHOLDING TU GRAZ GMBH, GRAZ, AT

Effective date: 20120924

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602006026266

Country of ref document: DE

Effective date: 20120910

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120731

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120318

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120726

REG Reference to a national code

Ref country code: AT

Ref legal event code: PC

Ref document number: 536423

Country of ref document: AT

Kind code of ref document: T

Owner name: TECHNISCHE UNIVERSITAET GRAZ, AT

Effective date: 20130724

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111207

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120726

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060726

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20140728

Year of fee payment: 9

Ref country code: DE

Payment date: 20140729

Year of fee payment: 9

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: AT

Payment date: 20140702

Year of fee payment: 9

Ref country code: FR

Payment date: 20140717

Year of fee payment: 9

Ref country code: SE

Payment date: 20140729

Year of fee payment: 9

Ref country code: GB

Payment date: 20140729

Year of fee payment: 9

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 602006026266

Country of ref document: DE

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: SE

Ref legal event code: EUG

REG Reference to a national code

Ref country code: AT

Ref legal event code: MM01

Ref document number: 536423

Country of ref document: AT

Kind code of ref document: T

Effective date: 20150726

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20150726

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160202

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20150731

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20150726

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20150731

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20160331

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20150726

Ref country code: SE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20150727

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20150731